For quantitative nucleic acid amplification, plasmids that contain copies of the target sequence(s) are routinely used as reference standards to generate calibration curves. Typically, a calibration curve is generated for each amplification target. A serial dilution of a known concentration of the calibrator nucleic acid is prepared, each diluted sample is subjected to PCR (or another amplified detection method), and the accumulation of product is monitored by detection of a signal (e.g., fluorescence) that accumulates in proportion to the amount of amplified target. The curve of accumulated signal from the amplified calibrator can then be used to determine the amount of the same target DNA in an unknown sample, i.e., by comparing the signal from the calibrators to the signal accumulated during amplification of the unknown. The amount of the target in the unknown sample can be calculated by comparison to the calibration data. See, e.g., Higuchi R, Fockler C, Dollinger G, Watson R, “Kinetic PCR Analysis: Real-time Monitoring of DNA Amplification Reactions”, Bio-Technology 11: 1026-1030 (1993).
Standard curve production becomes problematic when a multiplex detection format is used, i.e., when several targets are amplified in the same reaction. For such samples, generation of the calibration data requires use of different target nucleic acids that are provided in a mixture in precise, known ratios, e.g., mixtures in which there are the same number of copies of each of the different target nucleic acids. Producing such refined control mixtures can be problematic, however. For example, routine inaccuracies in the methods for measuring concentrations of different preparations of nucleic acid, e.g., due to co-purifying non-target nucleic acids, proteins, diverse extinction coefficients, and/or other solutes that influence instruments used to measure nucleic acids concentration, make it difficult to produce different target nucleic acid preparations that are precisely matched in concentration. The same inaccuracies make it difficult to reliably combine different nucleic acids to produce mixtures at known molar ratios, e.g., in equal molar amounts, for use as calibrators for multiplexed quantitative amplification reactions.
In addition the inaccuracies in concentration, the configurations and conformations of different target nucleic acids can affect the efficiency of amplification. For example, the same target sequence may produce slightly different quantitative amplification results depending on whether the target sequence is in a supercoiled plasmid, in large genomic DNA, or in short, fragmented DNA.